Alloantigen presentation by ethylcarbodiimide-treated dendritic cells induces T cell hyporesponsiveness, and prolongs organ graft survival

Ethylcarbodiimide (ECDI) couples soluble antigens (Ag) to lymphoid cells bestowing tolerizing potential. We examined whether ECDI-treated allogeneic dendritic cells (DC) could promote Ag-specific T cell unresponsiveness and prolong graft survival. Exposure of murine myeloid DC to ECDI did not affect surface immunophenotype but reduced their ability to cluster with T cells, enhanced their apoptotic death, and markedly reduced their allostimulatory activity. Anti-donor proliferative and cytotoxic T cell responses of mice primed with ECDI-treated DC were markedly inhibited. Secretion of both Th1 (IFNgamma) and Th2 cytokines (IL-5, IL-10) was suppressed. Cardiac allograft survival in mice preconditioned with a single injection of ECDI-DC was prolonged significantly. These results indicate that ECDI-treated DC promote T cell unresponsiveness to donor alloAgs and prolong transplant survival. The effects are not associated with sparing of Th2 responses, but may reflect inhibitory effects of apoptotic donor DC on host immune reactivity.     

Correlation of dendritic cell maturation and the formation of aggregates of poly-ubiquitinated proteins in the cytosol

Dendritic cells (DCs) are the most powerful antigen presenting cells (APCs) in the immune system. Therefore, they are able to take up antigen by phagocytosis, macropinocytosis or endocytosis, process it in the cytosol and present it to naive T cells. It is known that presentation of the immunodominant influenza virus nucleoprotein-derived CTL epitope is delayed in bone marrow-derived DCs (BMDCs) compared to non-professional APCs. This delay coincided with the formation of transient aggregations of ubiquitinated proteins (DALIS, dendritic cell aggresome-like induced structures), which contain probably defective ribosomal products (DRiPs). DRiPs appear in the cytosol of maturing DCs and macrophages. Normally, DRiPs are degraded rapidly by proteasomes. However, their storage in DALIS delays their degradation. So, it is hypothesized that DALIS can function as antigen depots allowing DCs to coordinate maturation and antigen presentation during their migration to the lymph nodes. Upon inhibition of several pathways among the in signal transduction pathways of DCs, like the phosphatidylinositol 3-kinase (PI3-K) or the mammalian target of Rapamycin (mTOR), the cells show a rendered maturation profile. The formation of DALIS is inhibited in these cells which can be expected to influence antigen processing and presentation. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 490, individual projects E6 to H.S. and E3 to R.H.).     

Polyphenol administration impairs T‐cell proliferation by imprinting a distinct dendritic cell maturational profile

Currently little is known as to how nutritionally derived compounds may affect dendritic cell (DC) maturation and potentially prevent inappropriate inflammatory responses that are characteristic of chronic inflammatory syndromes. Previous observations have demonstrated that two polyphenols quercetin and piperine delivered through reconstituted oil bodies (ROBs‐QP) can influence DC maturation in response to LPS leading to a modulated inflammatory response. In the present study, we examined the molecular effects of ROBs‐QP exposure on DC differentiation in mice and identified a unique molecular signature in response to LPS administration that potentially modulates DC maturation and activity in inflammatory conditions. Following LPS administration, ROBs‐QP‐exposed DCs expressed an altered molecular profile as compared with control DCs, including cytokine and chemokine production, chemokine receptor repertoire, and antigen presentation ability. In vivo ROBs‐QP administration suppresses antigen‐specific T‐cell division in the draining lymph nodes resulting from a reduced ability to create stable immunological synapse. Our data demonstrate that polyphenols exposure can drive DCs toward a new anti‐inflammatory molecular profile capable of dampening the inflammatory response, highlighting their potential as complementary nutritional approaches in the treatment of chronic inflammatory syndromes.     

Glucocorticoids down-regulate dendritic cell function in vitro and in vivo

Exogenous glucocorticoid hormones are widely used as therapeutical agents, whereas endogenous glucocorticoids may act as physiological immunosuppressants involved in the control of immune and inflammatory responses. The optimal activation of T lymphocytes requires two distinct signals: the major histocompatibility complex-restricted presentation of the antigen and an additional co-stimulatory signal provided by the antigen-presenting cells. There is ample evidence that, among the cells able to present the antigen, the dendritic cells (DC) have the unique property to activate antigen-specific, naive T cells in vitro and in vivo, and are therefore required for the induction of primary immune responses. In this work, we tested whether glucocorticoids affected the capacity of DC to sensitize naive T cells. Our data show that, in vitro, the steroid hormone analog dexamethasone (Dex) affects the viability of DC, selectively downregulates the expression of co-stimulatory molecules on viable DC, and strongly reduces their immunostimulatory properties. In vivo, a single injection of Dex results in impaired antigen presenting function, a finding which correlates with reduced numbers of splenic DC. These results show that glucocorticoids regulate DC maturation and immune function in vitro and in vivo and suggest that this mechanism may play a role in preventing overstimulation of the immune system.     

Different cytokines regulate antigen uptake and presentation of a precursor dendritic cell line

Langerhans cells (LC) and dendritic cells (DC) need to be activated in order to perform their antigen-presenting function. In this study, we explored the influence of cytokines on the uptake and presentation of protein antigens by the retrovirally immortalized myeloid cell line FSDC. This cell line was generated from mouse fetal skin and was previously shown to have the characteristics of early DC precursors. Both FSDC and bone marrow-derived DC (BM-DC) were more effective in the pinocytosis of FITC-conjugated ovalbumin (FITC-OVA) and dextran (FITC-DX) than B cells or macrophages. Pretreatment of FSDC with granulocyte/macrophage colony-stimulating factor (GM-CSF) ± interleukin (IL)-4 enhanced the pinocytic uptake of FITC-OVA and FITC-DX, but did not induce antigen-presenting capacity. In contrast, untreated FSDC or FSDC pre-incubated with GM-CSF ± IL-4 suppressed T cell responses. Treatment of FSDC with IFN-γ reduced pinocytosis but increased the expression of MHC and co-stimulatory/adhesion molecules and promoted efficient presentation of OVA protein or peptide to the specific DO11.10 T cell hybridoma or to naive CD4⁺ T cells from DO11.10 TCR-transgenic mice. The results suggest that antigen uptake and antigen presentation in DC are regulated by different cytokine signals provided by the surrounding tissue.     

Human and murine model cell lines for dendritic cell biology evaluated

Dendritic cells (DCs) are specialized antigen presenting cells that link innate and adaptive immune responses. As key mediators of T cell dependent immunity, DCs are considered primary targets for initiating immune responses in infectious diseases and cancer. Conversely, DCs can also play an important role in the induction of tolerance in organ transplantation, autoimmune disorders and allergy. While DCs have been used in clinical trials worldwide during the past decade, many of the highly specialized cell biological characteristics of DCs remain poorly understood. Small numbers of DCs can be isolated as terminally differentiated, post-mitotic cells form either blood or spleen. Alternatively, DC-precursors, such as monocytes or bone marrow-derived stem cells, can be isolated and differentiated into DCs in vitro. The relative low numbers of cells that can thus be obtained, combined with difficulties manipulating these terminally differentiated primary cells in vitro and in vivo, have seriously hampered studies aimed at exploring the cell biology of DCs. Good model cell lines therefore provide invaluable tools to study DC biology. So far most DC models are myeloid leukemia-derived cell lines that can be differentiated in vitro towards a DC phenotype. Here, we compared the phenotypical and functional characteristics of frequently used mouse and human DC-model cell lines. We conclude that, although none of these cell lines fully recapitulates all cell biological or immunological features of primary DCs, some of these cell lines provide valuable tools to study specific aspects of DC biology.     

A conditionally immortalized dendritic cell line which differentiates in contact with T cells or T cell-derived cytokines

A conditionally immortalized dendritic cell line was established from bone marrow of mice transgenic for a thermolabile mutant of the SV40 large T antigen under the control of the class I K^b promoter. At the permissive temperature of 33°–37°C, the line divides in the absence of granulocyte/macrophage colony stimulating factor. It shares a number of cell surface markers with bone marrow macrophages, but unlike macrophages, is constitutively major histocompatibility complex (MHC) class II⁺, negative for nonspecific esterase and unable to phagocytose sheep red blood cells. The cells show characteristic dendrites, an abundance of acidic vesicles and are highly active in endocytosis. If maintained at 33°C, the dendritic cell line processes and presents exogenous protein to MHC class II-restricted T cell hybrids and acts as potent mixed lymphocyte reaction stimulator, but fails to activate naive, resting T cells. Transfer to 39°C arrests growth and results in up-regulation of surface markers such as B7.1, CD40 and intercellular adhesion molecule-1. Further up-regulation of cell surface markers and acquisition of functional maturity occur following contact with T cells and their cognate antigen or in culture with a cytokine mixture derived from activated T cells.     

Induction of immunosuppressive functions of dendritic cells in vivo by CD4+CD25+ regulatory T cells: role of B7-H3 expression and antigen presentation

Suppressive functions of CD4+CD25+ regulatory T cells (Treg) are mainly studied by their interaction with conventional T cells. However, there is evidence that Treg also interact with antigen-presenting cells (APC), leading to suppression of APC function in in vitro coculture systems. Studying the in vivo distribution of Treg after injection, we found that Treg are located in direct proximity to dendritic cells (DC) and affect their functional maturation status. After contact to Treg, DC up-regulate the inhibitory B7-H3 molecule and display reduced numbers of MHC-peptide complexes, leading to impaired T cell stimulatory function. When Treg-exposed DC were used to immunize animals against antigens, the DC failed to produce a robust immune response as compared to control DC. Thus, these data indicate that Treg are able to inhibit DC activation and produce an inhibitory phenotype of DC. Accordingly, Treg may recruit DC for the amplification of immunosuppression by restraining their maturation in vivo and inducing an immunosuppressive phenotype of DC.     

Impaired in vivo CD4+ T cell expansion and differentiation in aged mice is not solely due to T cell defects: decreased stimulation by aged dendritic cells

CD4+ T cells regulate humoral and cell-mediated immune responses, which are progressively impaired in aging, resulting in susceptibility to infections and cancer. Dendritic cells (DCs) are major activators of T cells, providing signals that drive their expansion and differentiation. In this study, we asked if decreased CD4+ T cell responses were influenced by the age of DCs rather than being exclusively due to T cell defects. Old T cells transferred to young recipients expanded and differentiated similarly to young T cells. However, aged recipients were poor stimulators of both old and young T cells, which failed to acquire CD44 expression and produce interferon gamma (IFN-γ). DCs in aged hosts expressed fewer MHC-peptide complexes. The CD86 expression in the DCs of both hosts was similar; however, CD40 levels were reduced in old DCs. Finally, old DCs failed to produce inflammatory cytokines in response to LPS. Our results indicate that the impairment of aged CD4+ T cell function is intimately related to multiple alterations in aged DCs, rather than being caused solely by intrinsic T cell defects, suggesting that the function of aged T cells may be partially rescued in vivo when appropriate stimulation is applied. These findings are relevant to vaccination design for elderly populations.     

Control of cross-presentation during dendritic cell maturation

The initiation of most cytotoxic immune responses requires MHC class I-restricted presentation of internalized antigens to CD8(+) T lymphocytes, a process called cross-presentation. In dendritic cells (DC), the only antigen-presenting cells that activate naive T cells, cross-presentation is particularly efficient after internalization of opsonized antigens or immune complexes, which are cross-presented through a proteasome- and transporter associated with antigen processing (TAP)-dependent MHC class I antigen presentation pathway. We now show that FcgammaR-mediated cross-presentation is tightly regulated during DC maturation. Cross-presentation increases soon after activation by lipopolysaccharides, and it is then inhibited in fully mature cells. The initial induction of cross-presentation results from an increase of both antigen internalization and delivery to the cytosol, and from a slight rise in the activity of the proteasome and TAP. The subsequent block of cross-presentation in mature DC is a consequence of the selective down-modulation of antigen internalization and cytosolic delivery, while proteasome and TAP activities continue to rise. Therefore, FcgammaR-mediated cross-presentation is regulated during DC maturation by the selective control of antigen internalization and transport to the cytosol.     

巨细胞病毒抑制树突状细胞抗原递呈功能

目的： 探讨巨细胞病毒（CMV）对单核细胞来源的树突状细胞（DC）的感染效率及对受染细胞的功能影响。方法: 以50半数细胞培养感染量（TCID50）滴度的CMV与未成熟及成熟DC（imDC，mDC）共培养，逆转录－聚合酶链反应（RT-PCR）方法检测细胞内CMV即刻早期抗原（IEA）mRNA水平，间接免疫荧光技术检测受染细胞内早期抗原（EA）阳性率，流式细胞仪检测细胞胞内病毒晚期抗原pp65表达，BrdU ELISA法检测受染DCs（cmv-imDC,cmv-mDC）刺激异基因T细胞增殖能力。结果: 感染 12 h，cmv-mDC内IEA mRNA水平低于cmv-imDC，相对表达量分别为0.102±0.020和0.862±0.124（P<0.05）。24 h，imDC组EA阳性率高于mDC组，分别为(62.32±14.20)%和(10.78±3.04)%（P<0.01）。72 h，cmv-DC胞内低表达pp65抗原，imDC和mDC中阳性率分别为4.86%和0.82%。与未处理mDC相比，cmv-imDC经成熟诱导因子LPS作用后，其刺激异基因T细胞增殖能力较弱（均P<0.05）；而cmv-mDC，仅当DC/T细胞为 1∶1 时，刺激能力下降（P<0.05）。结论: CMV可有效感染imDC，并在细胞内复制活化；cmv-DC的抗原递呈能力下降。     

Pathways utilized by dendritic cells for binding, uptake, processing and presentation of antigens derived from HIV-1

The outcome following HIV infection depends on the nature and durability of the HIV-specific T cell response induced initially. The activation of protective T cell responses depends upon dendritic cells (DC), antigen-presenting cells which have the capacity to process and present viral antigens. DC pulsed with aldrithiol-2-inactivated HIV and delivered in vivo were reported to induce immune responses and promote virologic control in chronically HIV-1-infected subjects. To gain an understanding of this phenomenon, we characterized the steps involved in the presentation of antigens derived from aldrithiol-2-treated vs. infectious HIV-1 by DC. Antigen presentation, on both MHC class I and II, was independent of DC-specific ICAM-3-grabbing integrin, DEC-205 and macrophage mannose receptor, C-type lectins expressed by the DC. Inhibitor studies showed that presentation on MHC class I was dependent on viral fusion in a CD4/coreceptor-dependent manner, both at the cell surface and within endosomes, and access to the classical endosomal processing pathway. MHC class II presentation of HIV-associated antigens was dependent on active endocytosis, probably receptor-mediated, and subsequent degradation of virions in acidified endosomes in the DC. Our study brings forth new facts regarding the binding, uptake, and processing of chemically inactivated virions leading to efficient antigen presentation and should aid in the design of more effective HIV vaccines.     

Targeting dendritic cells to advance cross-presentation and vaccination outcomes

Dendritic cells (DCs) are a complex network of specialised antigen-presenting cells that are critical initiators of adaptive immunity. Targeting antigen directly to DCs in situ is a vaccination strategy that selectively delivers antigen to receptors expressed by DC subtypes. This approach exploits specific DC subset functions of antigen uptake and presentation. Here, we review DC-targeted vaccination strategies that are designed to elicit effective cross-presentation for CD8⁺ T cell immunity. In particular, we focus on approaches that exploit receptors highly expressed by mouse and human cDCs equipped with superior cross-presentation capacity. These receptors include DEC205, Clec9A and XCR1. Targeting DC receptors Clec12A, Clec4A4 and mannose receptor is also reviewed. Outcomes of DC-targeted vaccination in mouse models through to human clinical trials is discussed. This is a promising new vaccination approach capable of directly targeting the cross-presentation pathway for prevention and treatment of tumours and infectious diseases.     

Inclusion of Brefeldin A during dendritic cell isolation allows in vitro detection of cross-presented self-antigens

The mechanism of cross-presentation enables dendritic cells (DC) to induce immunity against intracellular pathogens and to tolerize autoreactive CD8 T cells. The antigen-presenting cells (APCs) responsible for cross-presentation of self-antigens have been identified as CD8⁺ CD11c⁺ DC. Isolation of these cells has been notoriously difficult, and the resulting responses of T cell hybridomas were too low to permit further studies. Here, we demonstrate that inclusion of Brefeldin A (BfA), an agent reported to block MHC class I–peptide complex turnover on the cell surface, during DC isolation from transgenic RIP-mOVA mice facilitated activation and proliferation of naïve OVA-specific CD8⁺ T cells in vitro. CD8⁺ DC were more efficient than CD8⁻ CD11c⁺. BfA also reversibly preserved expression of costimulatory molecules by DC, as evidenced by their expression of costimulatory markers and by an increased stimulatory capacity of DC matured in vivo by LPS. We conclude that the use of BfA notably improves sensitivity of detection of cross-presented self-antigens.     

Characteristics of antigen-independent and antigen-dependent interaction of dendritic cells with CD4+ T cells

Dendritic cells (DC) are the main antigen-presenting cells for the initiation of primary T cell-mediated immune responses. In the first stage of activation, T cells bind to DC in an antigen-independent manner. We studied the adhesion characteristics of human CD4⁺ T cells to DC generated from CD34⁺ hematopoietic progenitors following 12 to 13 days of culture in the presence of granulo-cyte/macrophage colony-stimulating factor and tumor necrosis factor-α. A majority of these cells had the morphology, phenotype and functions of DC. CD4⁺ T/DC adhesion was measured by means of fluorescence microscopy and flow cytometry. Four independent receptor/ligand pathways, LFA-1/ICAM, ICAM/LFA-1, CD2/LFA-3 and CD28/CD80, were involved in the transient adhesion of DC to CD4⁺ T cells in antigen-independent and specific alloantigen-dependent situations, as shown by blocking experiments using monoclonal antibodies. The antibodies also blocked a primary mixed lymphocyte reaction (MLR) in which DC were used as stimulatory cells. Adhesion of alloreactive CD4⁺ T cells to antigen-presenting DC was stronger than that of resting CD4⁺ T cells, while peak adhesion occurred after 5 and 20 min, respectively. The LFA-1 ligands involved in adhesion of resting CD4 T cells to DC and alloreactive CD4⁺ T cells to specific DC differed in part, since ICAM-3 on resting T cells and ICAM-1 on alloreactive T lymphocytes preferentially bound LFA-1. Studies of interactions between DC and phorbol ester-activated T cells expressing the CD40 ligand revealed a fifth independent adhesion pathway, CD40/CD40 ligand. CD4-mediated regulation of CD4⁺ T/DC adhesion was suggested by the observation that preincubation of CD4⁺ T cells and DC individually with anti-CD4 antibodies inhibited adhesion. In addition, antibodies specific for HLA class II molecules inhibited adhesion when used to pretreat DC but not alloactivated CD4⁺ T cells.     

正常妊娠中人外周血树突状细胞亚群的研究

目的：探讨髓样DC（MDC） 和浆细胞淋巴样DC（PDC）在正常妊娠中母胎免疫耐受机制中的作用。 方法： 选择正常妊娠妇女30例，分别在每个妊娠妇女早期、中期和晚期妊娠时采集外周血。应用流式细胞仪术，检测MDC和PDC占外周血单个核细胞的百分率及MDC/PDC比率，正常未妊娠女性为对照组。 结果： 与未妊娠妇女外周血中MDC和PDC的百分率（MDC, 0.32%±0.08%; PDC, 0.12%±0.05%）及MDC/PDC比率(2.96±1.39)相比：早期妊娠妇女外周血中MDC和PDC的百分率（MDC, 0.29%±0.07%; PDC, 0.11%±0.04%）及MDC/PDC比率(2.95±0.85)无显著差异（P>0.05）；中期妊娠妇女外周血中MDC和PDC的百分率（MDC, 0.11%±0.06%; PDC, 0.07%±0.03%）及MDC/PDC比率(1.52±0.44)降低（P<0.01）；晚期妊娠妇女外周血中MDC和PDC的百分率（MDC, 0.12%±0.06%; PDC, 0.08%±0.03%）及MDC/PDC比率(1.54±0.43)降低（P<0.01）。与正常早期妊娠妇女外周血中MDC和PDC的百分率及MDC/PDC比率相比，中、晚期妊娠显著降低（P<0.01）。 结论： 正常妊娠妇女中期和晚期妊娠时外周血中MDC和PDC的百分率及MDC/PDC比率均降低。     

Targeting antigen to bone marrow stromal cell‐2 expressed by conventional and plasmacytoid dendritic cells elicits efficient antigen presentation

Bone marrow stromal cell‐2 (BST‐2) has major roles in viral tethering and modulation of interferon production. Here we investigate BST‐2 as a receptor for the delivery of antigen to dendritic cells (DCs). We show that BST‐2 is expressed by a panel of mouse and human DC subsets, particularly under inflammatory conditions. The outcome of delivering antigen to BST‐2 expressed by steady state and activated plasmacytoid DC (pDC) or conventional CD8⁺ and CD8^? DCs was determined. T‐cell responses were measured for both MHC class I (MHCI) and MHC class II (MHCII) antigen presentation pathways in vitro. Delivering antigen via BST‐2 was compared with that via receptors DEC205 or Siglec‐H. We show that despite a higher antigen load and faster receptor internalisation, when antigen is delivered to steady state or activated pDC via BST‐2, BST‐2‐targeted activated conventional DCs present antigen more efficiently. Relative to DEC205, BST‐2 was inferior in its capacity to deliver antigen to the MHCI cross‐presentation pathway. In contrast, BST‐2 was superior to Siglec‐H at initiating either MHCI or MHCII antigen presentation. In summary, BST‐2 is a useful receptor to target with antigen, given its broad expression pattern and ability to access both MHCI and MHCII presentation pathways with relative efficiency.     

Split activity of interleukin-10 on antigen capture and antigen presentation by human dendritic cells: Definition of a maturative step

Human CD1⁺ CD14^- dendritic cells (DC) can be derived from CD14⁺ monocytes using granulocyte/monocyte colony-stimulating factor and interleukin (IL)-4. We have previously shown that IL-10 pre-treatment of such DC significantly inhibited their antigen-presenting capacity to CD4⁺ T cell clones. In this study, we further analyze how IL-10 influences antigen presentation. We first investigated whether IL-10 could alter the early stage of antigen presentation, the capture of antigen. This can be mediated by mannose receptor (MR)-mediated endocytosis and by fluid-phase uptake through macropinocytosis. IL-10-treated DC showed an enhancement of both mechanisms of antigen capture, as indicated by the increase of fluorescein isothiocyanate-dextran uptake through MR and lucifer yellow uptake. However, IL-10-treated DC, irradiated or glutaraldehyde-fixed, were less efficient than untreated DC in stimulating mixed leukocyte reaction as well as in inducing the activation of peptide-specific T cell clones, indicating that IL-10 achieves its effects mainly by modifying the cell surface phenotype of DC. HLA class I and II, as well as intercellular adhesion molecule (ICAM)-1, lymphocyte function-associated antigen-3, B7-1, B7-2 and ICAM-3 expression were either significantly increased or essentially unchanged, and the ability to bind the epitope recognized by the T cell clones was also unaffected regardless of IL-10 treatment. Our study also indicates that as-yet unidentified accessory molecules may play an essential role in T cell activation. Thus, the IL-10-treated DC possess an increased capacity to capture antigen, with a concomitant decreased stimulatory activity. Our study suggests that IL-10-treated DC have the characteristics of highly immature DC (high capture ability, low stimulatory potency) and may represent an early maturative step of human DC of monocytic origin.     

树突状细胞摄取和提呈颗粒化抗原的能力与颗粒载体的大小相关

目的：研究体外小鼠骨髓树突状细胞对2种不同大小bead-OVA复合物（0.04 μm bead和1.0μm bead）的摄取及class I途径抗原提呈能力。方法：以2h骨髓粘附细胞为前体细胞，用GM－CSF（1000U／ml）和IL－3（10ng/ml）培养5d，观察细胞对FITC标记的2种bead-OVA复合物的摄取，PMA、amiloride、cytochalasin D对摄取的抑制，以及细胞摄取后表达MHC分子和共刺激分子的情况，同时用OVA表位特异性T细胞杂交检测细胞摄取后通过class I途径活化CTL应答的能力。结果：树突状细胞对1．0μm bead-OVA的摄取明显高于对0．04μm bead-OVA，前者被上述3种抑制剂显著抑制，后者仅对amiloride和PMA抑制作用敏感，CCD无明显抑制作用。与摄取结果相反，0．04μm bead－OVA较1．0μm bead－OVA诱导更强的CD8细胞免疫应答，表型分析显示，细胞摄取0．04μm bead后，MHC分子和共刺激分子表达显著高于1．0μm的bead。结论：树突状细胞对2种bead的摄取能力和摄取机制不一样，0．04μm bead尽管摄取效率不如1．0μm bead，但通过class I途径提呈抗原的效率显著高于后者。     

囊泡运输相关蛋白VAP-33在小鼠树突状细胞肉瘤细胞株DCS细胞中的表达及其功能

目的 探讨囊泡运输相关蛋白VAP-33在小鼠树突状细胞肉瘤细胞株DCS细胞中的表达及其功能.方法 用1%TritonX-114提取DCS细胞膜蛋白,用已制备的DCS细胞多克隆抗体(pAb)及蛋白A+G琼脂糖进行免疫沉淀,所得样品利用质谱技术进行分析,检测到VAP-33蛋白在DCS细胞中有表达,继而DCS细胞经不同量抗原(150、850、1500μl)刺激24、48、72 h后观察细胞形态及吞噬能力的变化,同时通过间接免疫荧光、共聚焦显微镜、Western blot等方法检测了VAP-33的分布及表达变化.0.5 mol/L胰岛素刺激DCS细胞20 min后,采用Western blot检测DCS细胞全蛋白、胞质蛋白、膜蛋白中VAP-33、葡萄糖转运蛋白4(GLUT-4)的表达变化,共聚焦方法观察胰岛素刺激前后VAP-33和GLUT-4在DCS细胞中的表达及定位变化.实验中均以常规培养的DCS细胞作为对照.结果 VAP-33蛋白主要表达在DCS细胞膜和胞质中;在外来抗原刺激下,随抗原量的增加及作用时间的延长DCS细胞趋向成熟树突状细胞,VAP-33表达量降低,对辣根过氧化物酶的吞噬能力增强.胰岛素刺激后,VAP-33与GLUT-4有共定位.结论 VAP-33在树突状细胞来源的肿瘤细胞中表达,与树突状细胞的抗原加工有关,在葡萄糖的转运中起一定作用.